Our studies have addressed several major questions:[unreadable] [unreadable] 1) Expression of the CD25 antigen correlates with Treg function and Foxp3 expression in the mouse. The availability of a number of mAbs to human Foxp3 allowed us to perform a detailed analysis of the correlation between CD25 expression and Foxp3 expression. In contrast to our experience with mouse T cells, we found that only those CD4+ T cells that express CD25 at high levels are uniformly Foxp3+. A population of CD4+ T cells expressing intermediate levels of CD25 also contained some Foxp3+ T cells, but attempts to include these CD25int cells in our preparations always resulted in significant contamination with Foxp3- T cells. We purify human Foxp3+ T cells by first isolating all CD4+CD25+ T cells with magnetic beads and then use preparative cell sorting to obtain highly enriched (95% Foxp3+ T cells) Tregs. A number of assays have been used in the literature for measurement of the function of human Tregs in vitro. Some are modeled after the assays originally developed by us in the mouse and utilize responder cells, APC and soluble anti-CD3, while others prefer to use anti-CD3/CD28 beads to activate the responders in the absence of APC. As Treg suppression of mouse T cell activation induced by solid phase stimuli is remarkably inefficient, we routinely now use a culture system in which human CD4+CD25- T cells are activated with soluble OKT3 in the presence of T-depleted PBMC. [unreadable] [unreadable] 2) While it is clear the Foxp3 is the definitive marker for mouse Treg cells, several studies in man raised the possibility that Foxp3 can also be induced upon activation of human non-Treg cells. In some studies, these cells expressed a Treg phenotype, whereas in other studies the induction of Foxp3 did not correlate with a Treg phenotype. As our studies in the mouse clearly documented that stimulation of nave T cells in the presence of TGFbeta and IL-2 resulted in induction of Foxp3 expression and a complete Treg phenotype, we analyzed the effects of TGFbeta on the induction of Foxp3 on human T cells. When Foxp3- T cells were stimulated for 5 days with anti-CD3/CD28 and IL-2 in 5% autologous serum, 30% of the cells expressed Foxp3, while 80% expressed Foxp3 when TGFbeta was added to the cultures. When the cells were stimulated in the presence of anti-TGFbeta, less than 4% expressed Foxp3. Thus, human CD4+Foxp3- T cell resemble mouse cells in that Foxp3 expression can be induced by TGFbeta. However, the TGFbeta-induced cells lacked all the functional characteristics of real Treg, as they proliferated when stimulated via their TCR, failed to exert suppressor activity when co-cultured with freshly isolated CD4+CD25- T cells, and produced IL-2 and IFNgamma upon restimulation.[unreadable] [unreadable] 3) We have demonstrated that human Foxp3+ T cells, activated with plate-bound anti-hCD3, are almost as potent inhibitors of the proliferation of mouse CD4+Foxp3- responders (stimulated with soluble anti-mouse CD3 and mouse DC), as mouse CD4+Foxp3+ Treg. This result strongly suggests that at least in vitro a major component of the suppressive function of Foxp3+ Treg is preserved across the species. One advantage of this model is that it allows us to attempt to block the suppressive capacity of the human T cells with mAbs to human cell surface antigens that will not interfere with the activation of the mouse responder cells. We have tested a panel of several mAbs and noted that suppression by the human Tregs is completely reversed by anti-hCD11a (LFA-1), but not by anti-human CD54 (ICAM-1). We conclude from this that hCD11a on the human Tregs interacts with mouse CD54 on either the mouse responder T cell or DC, rather than with human CD54 on the human Treg. Human Tregs are fully competent suppressors of mouse CD4+Foxp3- T cells from CD54-/- mice in the presence of wild-type mouse DC indicating that one target of the human Tregs is the mouse DC rather that the mouse responder T cell. Thus, the suppressive effects of human Treg on mouse responder T cells closely mimic the potential function of Tregs cells in vivo where our recent studies have focused on the DC as the target.[unreadable] [unreadable] 4) The major obstacle for the use of human Tregs in cell-based therapy is the difficulty of obtaining a highly pure population after ex vivo expansion. A CD4+FOXP3+ population of > 90% purity can be isolated by FACS of the top 2-4% of CD4+ T cells with high CD25 expression (CD25hi) from peripheral blood, but frequently the percentage of FOXP3+ T cells decreases to 75% after one week and to 50% after two weeks of expansion by stimulation with anti-CD3/CD28 and IL-2. Furthermore, FACS-sorting of human T cells is not permitted under Good Manufacturing Practices (GMP) by US Food and Drug Administration. A more complex problem is the validity of FOXP3 as a bona fide marker of human Tregs. As TGFbeta is present in the serum used for cultures, an induction of FOXP3 expression in contaminating FOXP3 T cells may occur during expansion cultures of partially purified Tregs. While the expanded population might appear to be highly enriched in FOXP3+ cells, many of these cells may be induced FOXP3+ cells that lack Treg functions. We have identified two cell surface markers, latency associated peptide (LAP) and IL-1 receptor type II (CD121b) that are selectively expressed on activated Tregs, but not on activated CD4+FOXP3 or induced FOXP3+ cells. We have used these cell surface markers to design a protocol that allows for purification of FOXP3+ Tregs from ex vivo expansion cultures starting with leukapheresis preparations and using only magnetic bead targeting reagents. The final Treg product is composed of > 90% FOXP3+ cells that is highly anergic and suppressive in vitro. This method provides an important advance for the preparation of Tregs for cell-based immunotherapy to treat or prevent autoimmunity and transplant-related complications.